N. K. Blake

Enhanced ADP-glucose pyrophosphorylase activity in wheat endosperm increases seed yield

Yield in cereals is a function of seed number and weight; both parameters are largely controlled by seed sink strength. The allosteric enzyme ADP-glucose pyrophosphorylase (AGP) plays a key role in regulating starch biosynthesis in cereal seeds and is likely the most important determinant of seed sink strength. Plant AGPs are heterotetrameric, consisting of two large and two small subunits. We transformed wheat (Triticum aestivum L.) with a modified form of the maize (Zea mays L.) Shrunken2 gene (Sh2r6hs), which encodes an altered AGP large subunit. The altered large subunit gives rise to a maize AGP heterotetramer with decreased sensitivity to its negative allosteric effector, orthophosphate, and more stable interactions between large and small subunits. The Sh2r6hs transgene was still functional after five generations in wheat. Developing seeds from Sh2r6hs transgenic wheat exhibited increased AGP activity in the presence of a range of orthophosphate concentrations in vitro. Transgenic Sh2r6hs wheat lines produced on average 38% more seed weight per plant. Total plant biomass was increased by 31% in Sh2r6hs plants. Results indicate increased availability and utilization of resources in response to enhanced seed sink strength, increasing seed yield, and total plant biomass.

Genome comparisons reveal a dominant mechanism of chromosome number reduction in grasses and accelerated genome evolution in Triticeae

Single-nucleotide polymorphism was used in the construction of an expressed sequence tag map of Aegilops tauschii, the diploid source of the wheat D genome. Comparisons of the map with the rice and sorghum genome sequences revealed 50 inversions and translocations; 2, 8, and 40 were assigned respectively to the rice, sorghum, and Ae. tauschii lineages, showing greatly accelerated genome evolution in the large Triticeae genomes. The reduction of the basic chromosome number from 12 to 7 in the Triticeae has taken place by a process during which an entire chromosome is inserted by its telomeres into a break in the centromeric region of another chromosome. The original centromere–telomere polarity of the chromosome arms is maintained in the new chromosome. An intrachromosomal telomere–telomere fusion resulting in a pericentric translocation of a chromosome segment or an entire arm accompanied or preceded the chromosome insertion in some instances. Insertional dysploidy has been recorded in three grass subfamilies and appears to be the dominant mechanism of basic chromosome number reduction in grasses. A total of 64% and 66% of Ae. tauschii genes were syntenic with sorghum and rice genes, respectively. Synteny was reduced in the vicinity of the termini of modern Ae. tauschii chromosomes but not in the vicinity of the ancient termini embedded in the Ae. tauschii chromosomes, suggesting that the dependence of synteny erosion on gene location along the centromere–telomere axis either evolved recently in the Triticeae phylogenetic lineage or its evolution was recently accelerated.

Evaluation of "sequence-tagged-site" PCR products as molecular markers in wheat.

The polymerase chain reaction (PCR) is an attractive technique for many genome mapping and characterization projects. One PCR approach which has been evaluated involves the use of randomly amplified polymorphic DNA (RAPD). An alternative to RAPDs is the sequence-tagged-site (STS) approach, whereby PCR primers are designed from mapped low-copy-number sequences. In this study, we sequenced and designed primers from 22 wheat RFLP clones in addition to testing 15 primer sets that had been previously used to amplify DNA sequences in the barley genome. Our results indicated that most of the primers amplified sequences that mapped to the expected chromosomes in wheat. Additionally, 9 of 16 primer sets tested revealed polymorphisms among 20 hexaploid wheat genotypes when PCR products were digested with restriction enzymes. These results suggest that the STS-based PCR analysis will be useful for generation of informative molecular markers in hexaploid wheat.

Nucleotide diversity maps reveal variation in diversity among wheat genomes and chromosomes.

BackgroundA genome-wide assessment of nucleotide diversity in a polyploid species must minimize the inclusion of homoeologous sequences into diversity estimates and reliably allocate individual haplotypes into their respective genomes. The same requirements complicate the development and deployment of single nucleotide polymorphism (SNP) markers in polyploid species. We report here a strategy that satisfies these requirements and deploy it in the sequencing of genes in cultivated hexaploid wheat (Triticum aestivum, genomes AABBDD) and wild tetraploid wheat (Triticum turgidum ssp. dicoccoides, genomes AABB) from the putative site of wheat domestication in Turkey. Data are used to assess the distribution of diversity among and within wheat genomes and to develop a panel of SNP markers for polyploid wheat.ResultsNucleotide diversity was estimated in 2114 wheat genes and was similar between the A and B genomes and reduced in the D genome. Within a genome, diversity was diminished on some chromosomes. Low diversity was always accompanied by an excess of rare alleles. A total of 5,471 SNPs was discovered in 1791 wheat genes. Totals of 1,271, 1,218, and 2,203 SNPs were discovered in 488, 463, and 641 genes of wheat putative diploid ancestors, T. urartu, Aegilops speltoides, and Ae. tauschii, respectively. A public database containing genome-specific primers, SNPs, and other information was constructed. A total of 987 genes with nucleotide diversity estimated in one or more of the wheat genomes was placed on an Ae. tauschii genetic map, and the map was superimposed on wheat deletion-bin maps. The agreement between the maps was assessed.ConclusionsIn a young polyploid, exemplified by T. aestivum, ancestral species are the primary source of genetic diversity. Low effective recombination due to self-pollination and a genetic mechanism precluding homoeologous chromosome pairing during polyploid meiosis can lead to the loss of diversity from large chromosomal regions. The net effect of these factors in T. aestivum is large variation in diversity among genomes and chromosomes, which impacts the development of SNP markers and their practical utility. Accumulation of new mutations in older polyploid species, such as wild emmer, results in increased diversity and its more uniform distribution across the genome.

STS-PCR markers appropriate for wheat-barley introgression

Introgression of chromosomal segments across large taxonomic distances has long been an objective of scientists interested in understanding the relationships between genes and their effect on phenotype. Barley and wheat represent cultivated members of the Triticeae with different zones of adaptation, different responses to pathogens, and different end-use characteristics. Introduction of small, well-characterized chromosomal segments among grass relatives presents an opportunity to both better understand how genes perform in novel genomic environments and to learn more about the evolutionary novelties which differentiate related species. Since the distribution of the wheat-barley addition lines, the potential power and value of a comprehensive series of wheat/barley translocation lines has been widely appreciated. A scarcity of easy-touse markers which unambiguously distinguish barley loci from their wheat homologues has limited the ability of scientists to identify the relatively rare inter-chromosomal recombination events which are the necessary antecedents of these lines. Since the single most critical pathogen affecting U.S. wheat producers is Karnal bunt (Tilletia indica) and since barley carries a gene conferring immunity, molecular markers may prove practically and immediately important. In this report we describe a series of 135 barley-specific markers amplified by 115 primer sets developed from sequences from previously mapped restriction fragment length polymorphism (RFLP) markers. These easily distinguish the cognate barley products from their wheat counterparts and should find ready use in the identification of lines which contain wheat/barley translocation events.

Relationship of Dough Extensibility to Dough Strength in a Spring Wheat Cross

ABSTRACT A negative relationship between dough strength and dough extensibility would pose a problem for breeding hard wheats, as both dough strength and dough extensibility are desirable. We derived 77 recombinant inbred lines (RIL) from a cross between hard red spring wheat cultivars McNeal and Thatcher. McNeal produces flour with stronger dough and lower extensibility than does Thatcher. RIL were evaluated for strength-related properties using mixograph analysis and extensibility parameters using the Kieffer attachment to the TA.XT2 texture analyzer. Additionally, the RIL were test baked. Measurements using the mixograph and the Kieffer attachment were highly heritable. Maximum dough extensibility (Extmax) was negatively correlated with resistance to extension (Rmax) (r = -0.74) and with mixograph tolerance (r = -0.45). Loaf volume was correlated with both Rmax (r = 0.42) and area under the extensigraph curve (r = 0.44) based on partial correlation analysis adjusted for protein differences. Extmax was ...

Transformation ofGaeumannomyces graminis to benomyl resistance

SummaryGaeumannomyces graminis var.graminis andtritici were transformed to benomyl resistance using pBT3, a plasmid encoding fungicide-resistant β-tubulin. Either circular or linear plasmid DNA producedG. graminis var.graminis transformants in which plasmid DNA was integrated into the fungal genome. There was no evidence for autonomous plasmid replication in any of the transformants examined. 4/11 linear DNA transformants had a single plasmid copy, whereas 8/9 circular DNA transformants had multiple copies of the plasmid. Integration of transforming DNA occurred by nonhomologous recombination in all (20/20) of these transformants.

Multiple origins of allopolyploid Aegilops triuncialis

Abstract.Polyploidization is a key component of plant evolution. The number of independent origins of polyploid species traditionally has been underestimated. The objective of this study was to ascertain the number of origins of a tetraploid Aegilops species. We screened 84 primer sets to identify genome-specific primer sets for the tetraploid wheat relative [Aegilops triuncialis (UUCC genome)] and its diploid progenitors [Ae. umbellulata (UU genome) and Ae. caudata (CC genome)]. Primer sets G12 and G43 were U genome-specific and D21 was a C genome-specific primer. DNA sequence comparison of the G43 locus was used to estimate the number of polyploidization events in the formation of Ae. triuncialis. Parsimony analysis of G43 data revealed at least two independent formations of Ae. triuncialis. In the chloroplast hotspot region, located between genes rbcL and petA, sequence analysis suggested that at least three polyploidization origins might have occurred independently. Ae. triuncialis appears to be a tetraploid derived from multiple origins with minimal genome change after its formation.

The Isolation of fumB Mutants of Escherichia coli

Escherichia coli strains lacking the terminus region of the chromosome (min 29-36) due to an IS10-promoted deletion did not grow well in rich medium; they also did not grow on fumarate minimal medium because fumAC (min 35.7) is deleted. Strains with secondary mutations that partially suppress the deletion phenotype displayed healthier growth on rich medium and grew on minimal fumarate medium. These suppressor mutants had an IS10 insertion just upstream of the fumB structural gene (min 93.4). A strain with a Tn10 insertion at this location was constructed and used to delete nonessential fumB; fumB deletion mutants grew well on both rich and minimal fumarate media.

Phenotypic and Haplotype Diversity among Tetraploid and Hexaploid Wheat Accessions with Potentially Novel Insect Resistance Genes for Wheat Stem Sawfly

Haplotype diversity underlying Qss.msub.3BL solid‐stem QTL present among solid‐stem wheat landraces. Frequency and diversity of haplotypes vary among ploidy levels and geographical regions. New haplotypes potentially containing novel resistance alleles will augment WSS resistance. KASP markers can be used for marker assisted selection of the solid‐stem Qss.msub.3BL allele.